Application of corydaline in the preparation of shrimp hepatocellular carcinoma inhibitors

Abstract

The invention discloses the application of tetrahydropalmatine in preparing a shrimp hepatoenteric cytidylsis inhibitor, and discloses the application of tetrahydropalmatine in preparing a drug for preventing and / or treating shrimp hepatoenteric cytidylsis, a tetrahydropalmatine preparation for resisting shrimp hepatoenteric cytidylsis and a preparation method thereof. The tetrahydropalmatine preparation is characterized by being composed of the following raw materials and their mass percentages: 5-15% of tetrahydropalmatine, 3-6% of ethanol, 1-3% of Tween-20, 2-4% of water-soluble azone, and the rest being rapeseed oil. The preparation method comprises the following steps: weighing tetrahydropalmatine, Tween-20, water-soluble azone, ethanol and rapeseed oil for standby use; mixing tetrahydropalmatine, ethanol and rapeseed oil until tetrahydropalmatine is completely dissolved, then adding Tween-20 and water-soluble azone, and mixing to obtain the tetrahydropalmatine preparation. The tetrahydropalmatine preparation has the advantages of inhibiting the proliferation of shrimp hepatoenteric cytidylsis in shrimp and improving the weight gain rate of shrimp. The compound has low cost and simple use method.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of tetrahydropalmatine in the preparation of shrimp hepato-enterozoan inhibitors. Background Art

[0002] Shrimp Hepatocystis Enterocytozoon hepatopenaei , abbreviated as EHP) is one of the important pathogens of shrimp. Large-scale infection causes shrimp to have a decreased feeding capacity, slow growth or even stagnation, resulting in feed waste and easy to cause secondary bacterial diseases. It has the characteristics of wide distribution, strong transmission ability and host specificity. As an important component of new fishery drugs, Chinese herbal medicines have the advantages of low toxicity, low residue and high biosafety. Its powder or crude extract added to feed can improve the immunity of aquatic animals and increase resistance to pathogenic infection; and it is found that natural active monomers can accurately target pathogens, reduce or avoid the harm caused by the current use of agricultural and veterinary drugs, and has become one of the research hotspots of safe and green fishery prevention and control drugs.

[0003] Corydalis ethyl is derived from Corydalis yanhusuo, a plant of the Poppy family, and its structural formula is:

[0004]

[0005] Tetrahydropalmatine is also known as tetrahydropalmatine, with a molecular formula of C 21 H 25 NO4, molecular weight is 355.427, colorless or light yellow flake crystals, melting point is 147℃, easily soluble in ether, chloroform, soluble in ethanol, almost insoluble in water or alkaline solution. Currently, there is no research and application in the prevention of shrimp hepatoenteric cysts. Summary of the invention

[0006] One of the technical problems to be solved by the present invention is to provide an application of tetrahydropalmatine in the preparation of shrimp hepatoenteric cytozoon inhibitors; the second is to provide an application of tetrahydropalmatine in the preparation of drugs for preventing and / or treating shrimp hepatoenteric cytozoon disease; the third is to provide a tetrahydropalmatine preparation for inhibiting shrimp hepatoenteric cytozoon and a preparation method thereof, wherein the preparation can effectively inhibit the replication of EHP in shrimp.

[0007] The technical solution adopted by the present invention to solve the above technical problem is: application of tetrahydropalmatine in the preparation of shrimp hepatoenteric cytozoon inhibitor.

[0008] The present invention also provides an application of tetrahydropalmatine in preparing a medicine for preventing and / or treating shrimp hepatoenteric cytosis.

[0009] The invention also provides a tetrahydropalmatine preparation for inhibiting shrimp hepatoenteric cysts, which is composed of the following raw materials and their mass percentages: 5-15% tetrahydropalmatine, 3-6% ethanol, 1-3% Tween-20 and 2-4% water-soluble azone, and the rest is rapeseed oil.

[0010] Preferably, the tetrahydropalmatine preparation is composed of the following raw materials and their mass percentages: 15% tetrahydropalmatine, 6% ethanol, 72% rapeseed oil, 3% Tween-20 and 4% water-soluble azone.

[0011] Preferably, the tetrahydropalmatine preparation is composed of the following raw materials and their mass percentages: 15% tetrahydropalmatine, 6% ethanol, 74% rapeseed oil, 2% Tween-20 and 3% water-soluble azone.

[0012] Preferably, the tetrahydropalmatine preparation is composed of the following raw materials and their mass percentages: tetrahydropalmatine 15.0%, ethanol 6.0%, rapeseed oil 79.0%, Tween-20 1.0% and water-soluble azone 2.0%.

[0013] The present invention also provides a method for preparing the tetrahydropalmatine preparation for inhibiting shrimp hepatoenteric cysts, comprising the following steps: weighing tetrahydropalmatine, Tween-20, water-soluble azone, ethanol and rapeseed oil for later use; mixing the tetrahydropalmatine, ethanol and rapeseed oil until the tetrahydropalmatine is completely dissolved, then adding Tween-20 and water-soluble azone, and mixing to obtain the tetrahydropalmatine preparation.

[0014] The present invention also provides the use of the tetrahydropalmatine preparation for inhibiting shrimp hepatoenteric cysts in shrimp farming, wherein the tetrahydropalmatine preparation is added to shrimp farming water at a concentration of 40 mg / L.

[0015] Compared with the prior art, the advantages of the present invention are as follows: the present invention discloses for the first time an application of tetrahydropalmatine in the preparation of shrimp hepatoenteric cytia inhibitors and drugs for preventing and / or treating shrimp hepatoenteric cytia disease; tetrahydropalmatine, as an effective ingredient of a new type of medicament, has low toxicity, is not prone to drug resistance, and can effectively prevent and treat shrimp hepatoenteric cytia disease; and further discloses a tetrahydropalmatine preparation; as a new type of fishery medicine, the tetrahydropalmatine preparation is natural and pollution-free, is not prone to drug resistance in shrimp, can effectively prevent and treat shrimp hepatoenteric cytia disease; the preparation has low cost, is simple to use, and is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The toxicity and anti-EHP activity analysis results of tetrahydropalmatine, where A is the survival rate of healthy shrimp under different concentrations of tetrahydropalmatine, and B is the EHP copy number in diseased shrimp under different concentrations of tetrahydropalmatine;

[0017] Figure 2is the preventive effect of tetrahydropalmatine on EHP, wherein A is the determination result of the EHP copy number of healthy shrimps infected with EHP after being pretreated with tetrahydropalmatine, and B is the determination result of the weight of healthy shrimps infected with EHP after being pretreated with tetrahydropalmatine;

[0018] Figure 3 is the therapeutic effect of tetrahydropalmatine on EHP, wherein A is the result of measuring the EHP copy number of healthy shrimp after being infected with EHP and then treated with tetrahydropalmatine, and B is the result of measuring the weight of healthy shrimp after being infected with EHP and then treated with tetrahydropalmatine;

[0019] Figure 4 These are the results of the effect of ferroptosis on the horizontal transmission of EHP when healthy and diseased shrimps coexist. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to the accompanying drawings.

[0021] Specific Example 1: Analysis of the anti-EHP activity of tetrahydropalmatine.

[0022] 1. Experimental materials: Tetrahydropalmatine, dimethyl sulfoxide (DMSO), and anhydrous ethanol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and EHP and Penaeus vannamei were provided by Zhejiang Marine Aquaculture Research Institute. Preparation of Tetrahydropalmatine stock solution: Weigh Tetrahydropalmatine and fully dissolve it in DMSO to prepare a stock solution with a final concentration of 50,000 mg / L and store it at 4°C for later use.

[0023] 2. EHP copy number detection: Use the marine animal tissue genomic DNA rapid extraction kit to extract shrimp hepatopancreas DNA, and use an ultra-micro spectrophotometer to detect DNA concentration (adjust the final concentration to 100 ng / μL) and purity before conducting qPCR amplification experiments. qPCR amplification primers include ENF 185 Forward and reverse amplification primers, ENF 185 The nucleotide sequence of the forward amplification primer is shown in SEQ ID NO.1: 5'-GTAGCGGAACGGATAGGG-3', ENF 185The nucleotide sequence of the reverse amplification primer is shown in SEQ ID NO.2: 5'-CCAGCATTGTCGGCATAG-3', and the qPCR amplification reaction system is as follows: 2 × Es Taq MasterMix 7.5μL, 10 μM forward amplification primer 0.3 μL, 10 μM reverse amplification primer 0.3 μL, template DNA 1μL, ddH2O 5.9 μL. The qPCR amplification reaction program is as follows: 95℃5 min; 95℃ 30 s, 55.5℃ 30 s, 72℃ 30 s, 35 cycles; 72℃ 10 min. According to pMD19T-ENF 185 The standard curve made from the standard product was used to convert the qPCR results to obtain the EHP copy number.

[0024] 3. Tetrahydropalmatine toxicity test: Healthy shrimps were divided into 5 groups, 15 shrimps in each group. The experiment was conducted by immersion. The concentrations of Tetrahydropalmatine in the aquaculture water were 5, 10, 20, 40 and 80 mg / L, respectively. The control group was a DMSO concentration of 0.02% in the aquaculture water. The survival rate of shrimps was counted every 24 hours, and the safe concentration of Tetrahydropalmatine was determined based on the survival rate of shrimps.

[0025] The results are as follows Figure 1 As shown in Figure A, compared with the control group (the concentration of tetrahydropalmatine was 0), when the concentration of tetrahydropalmatine was 20, 40 and 80 mg / L, the survival rate of shrimp was significantly reduced; while when the concentration of tetrahydropalmatine was 5 and 10 mg / L, the survival rate of shrimp was close to 100%, and there was no significant difference between the two. In summary, 10 mg / L of tetrahydropalmatine was selected as a safe concentration for subsequent experiments.

[0026] 4. Detection of the anti-EHP activity of tetrahydropalmatine: Four groups of culture water were added with tetrahydropalmatine at concentrations of 1.25 mg / L, 2.5 mg / L, 5 mg / L and 10 mg / L, respectively. Each group was fed with fresh hepatopancreas of EHP-infected shrimp (the EHP concentration in the fresh hepatopancreas of EHP-infected shrimp was 1.6×10 6 The control group was fed with fresh hepatopancreas of EHP-infected shrimp in aquaculture water containing 0.02wt% DMSO, and the feeding amount was 2 g / 10 shrimp. Each group of 10 shrimps was sampled on the 7th day of infection and the EHP copy number was detected by qPCR.

[0027] The results are as follows Figure 1As shown in B, the concentration of tetrahydropalmatine is correlated with the number of EHP copies. Compared with the control group, the number of EHP copies in shrimp decreased significantly when the concentration of tetrahydropalmatine was 5 and 10 mg / L; while it decreased slightly when the concentration of tetrahydropalmatine was 1.25 and 2.5 mg / L, but it was not significant. These results show that tetrahydropalmatine can significantly inhibit the proliferation of EHP at 10 mg / L.

[0028] 5. Preventive effect of tetrahydropalmatine on EHP: Healthy shrimps were divided into two groups, 30 shrimps in each group. The culture water of the experimental group contained 10 mg / L tetrahydropalmatine, and the feed was fed three times a day. The culture water containing 10 mg / L tetrahydropalmatine was replaced every 3 days, which was recorded as EHP. 延胡索乙素 The culture water of the control group contained 0.02% DMSO, recorded as EHP DMSO , feed was given three times a day, and the culture water containing DMSO was changed every 3 days. After 15 days, both groups of shrimp were fed with fresh hepatopancreas of EHP-infected shrimp at a feeding amount of 2 g / 10 shrimps, and samples were collected on the 10th, 20th and 30th days after EHP infection, and the EHP copy number was detected by qPCR. The shrimps of both groups were weighed on the 30th day.

[0029] The results are as follows Figure 2 As shown in Figure A, after shrimp were pretreated with corydalis, they were then infected with EHP. At 10 and 20 days after infection, the number of EHP copies in the experimental group was significantly lower than that in the control group. At 30 days after infection, the number of EHP copies in the experimental group was lower than that in the control group, but there was no significant difference. Figure 2 As shown in B, on the 30th day after infection, the shrimps in the experimental group and the control group were weighed, and it was found that the weight of the shrimps in the experimental group increased, with a weight gain rate of 12%, but there was no significant difference. These results indicate that tetrahydropalmatine has a certain preventive effect on EHP.

[0030] 6. Therapeutic effect of tetrahydropalmatine: Healthy shrimp were fed with fresh hepatopancreas of EHP-infected shrimp at a feeding rate of 2 g / 10 shrimps and divided into two groups, with 30 shrimps in each group. The culture water of the experimental group contained 10 mg / L tetrahydropalmatine and the feed was fed three times a day. The culture water containing 10 mg / L tetrahydropalmatine was replaced every 3 days. The culture water of the control group contained 0.02% DMSO, recorded as EHP DMSO Feed was given three times a day, and the culture water containing DMSO was changed every 3 days. Samples were taken on the 10th, 20th and 30th days to detect the number of EHP copies in the shrimp, and the weight of each group of shrimp was measured on the 30th day.

[0031] The results are as follows Figure 3As shown in Figure A, after shrimp were infected with EHP, they were treated with tetrahydropalmatine. On the 10th, 20th and 30th days after treatment, the EHP copy number in the shrimp of the control group and the experimental group showed significant differences. Among them, after 30 days of treatment, the inhibition rate of EHP copy number was 81%. Figure 3 As shown in Figure B, in terms of body weight, the shrimp in the tetrahydropalmatine-treated group were significantly heavier than those in the control group, with a weight gain rate of 20%. These results indicate that tetrahydropalmatine has a good therapeutic effect on EHP.

[0032] 7. Effect of corydalis on horizontal transmission of EHP: Healthy shrimp were fed with fresh hepatopancreas of EHP-infected shrimp at a feeding rate of 2 g / 10 shrimp. After 7 days, the EHP-infected shrimp were divided into two groups, with 15 shrimp in each group. At the same time, 15 healthy shrimp were placed in each group, and the sick shrimp and healthy shrimp were separated by gauze. The culture water of the experimental group contained 10 mg / L corydalis, and the culture water of the control group contained 0.02wt% DMSO. On the 10th, 20th and 30th days after cohabitation, healthy shrimp were taken to detect the number of EHP copies in the body.

[0033] The results are as follows Figure 4 As shown, tetrahydropalmatine can significantly inhibit the horizontal spread of EHP. Compared with the control group, when tetrahydropalmatine was present, the inhibition rates of EHP copy numbers in healthy shrimp were 48%, 47% and 44% at the 10th, 20th and 30th days of cohabitation, respectively. These results indicate that tetrahydropalmatine has the potential to prevent EHP outbreaks.

[0034] Specific Example 2: Preparation of a tetrahydropalmatine preparation for inhibiting shrimp hepatoenteric cytozoa.

[0035] The sources of tetrahydropalmatine, Litopenaeus vannamei and EHP are the same as those in the above specific embodiment 1.

[0036] 1. Solvent selection for tetrahydropalmatine preparation: By observing and comparing the solubility of tetrahydropalmatine in rapeseed oil, peanut oil, ethanol and acetic acid solvents, it was found that tetrahydropalmatine had no stratification and precipitation in rapeseed oil and ethanol, and the dissolution effect was good. Rapeseed oil and ethanol were combined in different proportions, and the following 8 groups of experiments were set up to determine the solvent ratio with high solubility and safety.

[0037] Experimental group 1: Weigh 15.0 g of tetrahydropalmatine, 6.0 g of ethanol, and 72.0 g of rapeseed oil, mix them until tetrahydropalmatine is completely dissolved, then add 3.0 g of surfactant Tween-20 and 4.0 g of transdermal water-soluble azone, mix well to obtain tetrahydropalmatine preparation.

[0038] Experimental group 2: Weigh 13.0 g of tetrahydropalmatine, 5.0 g of ethanol, 76.0 g of rapeseed oil, 3.0 g of Tween-20 and 3.0 g of water-soluble azone, mix thoroughly to obtain tetrahydropalmatine preparation, and the rest is the same as experimental group 1.

[0039] Experimental group 3: Weigh 10.0 g of tetrahydropalmatine, 4.0 g of ethanol, 81.0 g of rapeseed oil, 3.0 g of Tween-20 and 2.0 g of water-soluble azone, mix thoroughly to obtain tetrahydropalmatine preparation, and the rest is the same as experimental group 1.

[0040] Experimental group 4: 9.0 g of tetrahydropalmatine, 3.0 g of ethanol, 84.0 g of rapeseed oil, 2.0 g of Tween-20 and 2.0 g of water-soluble azone were weighed and mixed thoroughly to obtain tetrahydropalmatine preparation. The rest was the same as that of experimental group 1.

[0041] Experimental group 5: Weigh 7.0 g of tetrahydropalmatine, 4.0 g of ethanol, 86.0 g of rapeseed oil, 1.0 g of Tween-20 and 2.0 g of water-soluble azone, mix thoroughly to obtain tetrahydropalmatine preparation, and the rest is the same as experimental group 1.

[0042] Experimental group 6: Weigh 5.0 g of tetrahydropalmatine, 3.0 g of ethanol, 85.0 g of rapeseed oil, 3.0 g of Tween-20 and 4.0 g of water-soluble azone, mix them thoroughly to obtain tetrahydropalmatine preparation, and the rest is the same as experimental group 1.

[0043] Table 1 Results of different solvent ratios of tetrahydropalmatine preparations

[0044]

[0045] As shown in Table 1, the solubility of tetrahydropalmatine in the solvent composed of ethanol and rapeseed oil (mass ratio range is 1:12-1:28) in experimental group 1 is the highest, and its solubility exceeds 15 g.

[0046] 2. Anti-EHP activity of excipients of corydalis preparations: This experiment compared the following combinations to determine the effects of other substances (solvents, Tween-20 and water-soluble azone) in corydalis preparations on the anti-EHP activity of corydalis. Healthy shrimp were randomly placed in a culture bucket (30 L culture water / bucket, 15 shrimp / bucket), and corydalis preparations of the following experimental groups (total mass of 2 g) and fresh hepatopancreas of EHP-infected shrimp were added to the culture water (the EHP concentration in the fresh hepatopancreas of EHP-infected shrimp was 1.6×10 6 The feeding amount was 2 g / 10 shrimps, and the EHP copy number in shrimps was detected after 7 days of incubation.

[0047] Experimental group 1: 15.0 g of tetrahydropalmatine, 6.0 g of ethanol and 79.0 g of rapeseed oil; Experimental group 2: 15.0 g of tetrahydropalmatine, 6.0 g of ethanol, 72.0 g of rapeseed oil, 3.0 g of Tween-20 and 4.0 g of water-soluble nitrogen; Experimental group 3: 15.0 g of tetrahydropalmatine, 6.0 g of ethanol, 74.0 g of rapeseed oil, 2.0 g of Tween-20 and 3.0 g of water-soluble nitrogen; Experimental group 4: 15.0 g of tetrahydropalmatine, 6.0 g of ethanol, 79.0 g of rapeseed oil, 1.0 g of Tween-20 and 2.0 g of water-soluble nitrogen; Experimental group 5: 6.0 g of ethanol and 94.0 g of rapeseed oil. The experimental results are shown in Table 2 below.

[0048] Table 2 Anti-EHP activity results of different formulations of tetrahydropalmatine preparations

[0049]

[0050] As shown in Table 2, experimental group 5 has no significant anti-EHP activity, experimental group 2 and experimental group 3 have the best anti-EHP activity, followed by experimental group 1 and experimental group 4. The above results show that the anti-EHP activity of the transdermal water-soluble azone and the surfactant Tween-20 can be improved when the total mass proportion of the transdermal water-soluble azone and the surfactant Tween-20 in the tetrahydropalmatine preparation is less than or equal to 7%.

[0051] 3. Anti-EHP activity of corydalis preparations at different added concentrations: Healthy shrimps were randomly placed in culture barrels (30 L culture water / barrel, 15 shrimps / barrel). The experiment set up four groups of culture water with concentrations of 0 mg / L, 10 mg / L, 20 mg / L and 40 mg / L of corydalis preparations (corydalis 15.0 g, ethanol 6.0 g, rapeseed oil 72.0 g, Tween-20 3.0 g and water-soluble nitrogen 4.0 g). Each group was fed with fresh hepatopancreas of EHP-infected shrimps at a feeding amount of 2 g / 10 shrimps. The water containing the same concentration of corydalis preparations was replaced every 3 days. The hepatopancreas of shrimps in each group was collected on the 30th day to detect the number of EHP copies; at the same time, 10 shrimps were taken from each group, weighed and compared. The results are shown in Table 3 below.

[0052] Table 3 Anti-EHP activity of tetrahydropalmatine preparations

[0053]

[0054] As shown in Table 3, adding different doses of tetrahydropalmatine preparations to the aquaculture water can inhibit the replication of EHP in shrimp and promote its weight gain. When the addition amount of tetrahydropalmatine preparations was 40 mg / L, the inhibition rate of EHP replication in shrimp was 78%, and the weight of shrimp increased significantly compared with the control group. In summary, tetrahydropalmatine preparations can effectively prevent and treat EHP infection.

[0055] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. An application of tetrahydropalmatine in the preparation of shrimp hepatoenteric cytozoon inhibitors.

2. Use of tetrahydropalmatine in the preparation of drugs for preventing and / or treating shrimp hepatoenterozoan disease.

3. The use of tetrahydropalmatine according to claim 2 in the preparation of a drug for preventing and / or treating shrimp hepatoenteritis cytosis, characterized in that The drug is a tetrahydropalmatine preparation, which is composed of the following raw materials and their mass percentages: 5-15% tetrahydropalmatine, 3-6% ethanol, 1-3% Tween-20, 2-4% water-soluble azone, and the rest is rapeseed oil.

4. The use of tetrahydropalmatine according to claim 3 in the preparation of a drug for preventing and / or treating shrimp hepatoenteritis cytosis, characterized in that The tetrahydropalmatine preparation is composed of the following raw materials and their mass percentages: 15.0% tetrahydropalmatine, 6.0% ethanol, 72.0% rapeseed oil, 3.0% Tween-20 and 4.0% water-soluble azone.

5. The use of tetrahydropalmatine according to claim 3 in the preparation of a drug for preventing and / or treating shrimp hepatoenteritis cytosis, characterized in that The tetrahydropalmatine preparation is composed of the following raw materials and their mass percentages: 15.0% tetrahydropalmatine, 6.0% ethanol, 74.0% rapeseed oil, 2.0% Tween-20 and 3.0% water-soluble azone.

6. The use of tetrahydropalmatine according to claim 3 in the preparation of a drug for preventing and / or treating shrimp hepatoenteritis cytosis, characterized in that The preparation method of the tetrahydropalmatine preparation comprises the following steps: weighing tetrahydropalmatine, Tween-20, water-soluble azone, ethanol and rapeseed oil for later use; mixing tetrahydropalmatine, ethanol and rapeseed oil until the tetrahydropalmatine is completely dissolved, then adding Tween-20 and water-soluble azone, and mixing to obtain the tetrahydropalmatine preparation.

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